Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J. Cell Biol.142:923-936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.

An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.

To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

A membrane-associated protein complex with selective binding to the clathrin coat adaptor AP1.

Adaptors are the membrane-binding components of clathrin-coated vesicles. The interaction of the trans-Golgi coat adaptor AP1 with membrane-associated proteins was analyzed by affinity chromatography. Proteins of 83 and 52 kDa bound specifically to the core domain of AP1 and showed no interaction with AP2 or other clathrin-coated vesicle proteins. The AP1-binding proteins were tightly membrane-associated, though behaved as peripheral membrane proteins. They were detected in membranes depleted of clathrin-coated vesicles and not in coated vesicles, suggesting that the interaction of these proteins with AP1 may precede coated vesicle budding. Co-fractionation of the AP1-binding proteins with trans-Golgi network membrane was also observed. Upon gel filtration, both AP1-binding proteins eluted in a high molecular mass complex which was labile at high concentrations of Tris. The 83 kDa protein bound to AP1 affinity resin in the absence of the 52 kDa protein. In contrast, the separated 52 kDa protein did not bind AP1, suggesting that the 83 kDa protein is the AP1-binding component of the complex. Characterization of this protein complex defines a novel membrane-associated component that specifically interacts with AP1 and may contribute to its function in forming clathrin-coated vesicles.

Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

Activation of (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to diolepoxides by human polymorphonuclear leukocytes or myeloperoxidase.

Previous studies have demonstrated that the interaction of (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(+-)-B[a]P-7,8-diol] with 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human polymorphonuclear leukocytes (PMNs) elicited genotoxic effects in bacteria and mammalian cells. Structure-activity studies with various polycyclic aromatic hydrocarbon derivatives suggest that a diolepoxide intermediate(s) was being formed from this chemical-cell interaction. In this study, we demonstrate by stereochemical analysis of tetraol products that primarily anti-diolepoxides are being formed from (+-)-B[a]P-7,8-diol by TPA-stimulated PMNs with an anti/syn ratio of 6. Likewise, a myeloperoxidase (MPO)-H2O2 system generated primarily anti-diolepoxides of B[a]P-7,8-diol with an anti/syn ratio greater than 5. Such ratios are indicative of the epoxidation of B[a]P-7,8-diol via a peroxyl radical or a ferryl oxygen transfer-mediated reaction. Addition of azide, an MPO inhibitor, resulted in decreased tetraols from B[a]P-7,8-diol by PMNs or the MPO system. These studies further support the concept that the activation of B[a]P-7,8-diol by PMNs could create a highly localized genotoxic environment which could impact on human health.

Internalization of the Hm1 muscarinic cholinergic receptor involves the third cytoplasmic loop.

The m1 muscarinic receptor was previously shown to stimulate phosphatidyl inositol (PI) turnover and to internalize rapidly upon agonist activation. Three receptor mutants with large deletions of the third cytoplasmic loop (i3) of human Hm1, leaving only 11 and 8 amino acids at the amino and carboxy terminal junctions of i3, respectively, retained full ability to stimulate PI turnover, when expressed in U293 cells, but receptor internalization was greatly reduced in two mutants with deletions reaching close to the NH2 terminal of i3. We propose that a receptor domain located toward the amino terminal junction of i3 plays a role in Hm1 internalization.